Membrane preparation and process for separating the dissolved and undissolved constituents of milk

ABSTRACT

In a process for separating the dissolved and undissolved constituents of milk, a microporous membrane with a pore size in the range of 0.1 to 2 μm is pretreated with an aqueous solution, dispersion or emulsion of lipids or peptides and the milk is separated on the pretreated membrane.

The invention concerns a process for separating the dissolved andundissolved constituents of milk.

The composition of the milk of mammals is well known. The dry content ofthe cow milk is know to be about 12.7% on average, of which 3.7% are fatcomponents, 3.4% total serum protein. 4.7% lactose and about 0.7% ash.The protein component mainly consists of caseins and whey proteins.Moreover there are a non-proteinaceous nitrogen fraction, proteasepeptones and minor proteins, which are mainly enzymes.

Ordinarily milk is separated into caseins and whey proteins. Thisseparation is by means of the so-called rennet precipitation in whichrennin is added to warmed-up milk (30°-35° C.). The caseins thenprecipitate, the whey proteins remaining in solution. The same appliesto the so-called acid precipitation of the caseins which takes place atthe iso-electric point (cow milk pH 4.7). Caseins are heat-resistant,whereas the whey proteins are thermally labile.

It is further known to concentrate milk by reverse osmosis, in whichpractically pure water is removed from the milk, i.e. all the dissolvedand undissolved milk constituents including the salts dissolved in themilk remain in the retentate and the permeate essentially consists ofpure water. This is the way essentially in which presently the so-calledcondensed milk is prepared. However, reverse osmosis also is used toconcentrate whey and when making curd-, yogurt-, and sour-milk.

It is further known to treat milk with ultra-filtration. However, onlywhen using skim milk or whey will there be absence of difficulties inthe enrichment of the milk proteins. Ultrafiltration of nativefull-cream milk, on the other hand, results only in incompleteseparation. Milk separation by means of conventional membrane filters isimpossible.

It is comparatively expensive to secure the whey proteins, which areespecially desirable for nutritional and physiological reasons, whenusing the known methods. Moreover, the whey proteins obtained using theconventional methods as a rule are bacteria-contaminated and containfat, and further contain the calcium and phosphate ions released uponthe separation into caseins and whey proteins.

Accordingly, the object of the invention is to provide an improvedprocess for separating milk into dissolved and undissolved constituentsby resorting to membranes.

It was found in surprising manner that milk can be separated into itsdissolved and undissolved constituents if it is made to pass through amicroporous membrane pretreated with lipids and/or peptides.

Accordingly, the object of the invention is a process for separating thedissolved and undissolved milk ingredients, which is characterized bypretreating a micro-porous membrane with a pore size in the range from0.1 to 2 microns (u) with lipids and/or peptides, and by separating themilk at the membrane so pretreated.

Advantageously, a fluorocarbon polymer based membrane shall be used.Especially preferred is a polyvinylidene fluoride membrane, for instancea so called DURAPORE membrane made by Millipore Co. (for instance theGLVP or HVLP type).

The membrane's pore sizes are preferably in the range from 0.1 to 1 u,in particular between 0.1 and 0.6 u.

The pretreatment may be with lipids or peptides alone, first with lipidsand then with peptides. By means of this pretreatment, a membrane isobtained, which--contrary to the case of an untreated membrane--allowsseparating the milk into its dissolved and undissolved constituents. Noexplanation for the effect of pretreatment can be given. Still it mightbe a polarization effect.

The pretreatment of the membrane is simply carried out by exposing it toa flowing solution, emulsion or dispersion of the lipids and/or peptidesin water. As a rule, lipid emulsion of 0.1 to 10%, preferably from 2 to5% or a peptide solution of 0.01 to 10%, preferably from 0.1 to 5% shallsuffice.

Appropriately, the pretreatment takes place at a temperature of 8° to40° C. in particular from 15° to 35° C.

The duration of the pretreatment depends on the particular lipid orpeptide used and on the concentration of the emulsion or solutionflowing through the membrane. As a rule, pretreatment of 1 to 8 hours(h), in particular 2 to 5 h, is adequate to obtain a membrane permittingmilk separation.

Especially for membranes with low pore sizes (for instance 0.1 to 0.4u). The desired effect can be obtained with lipids alone. When membranesof larger pore sizes are pretreated with lipids, the milk constituentscannot be fully separated. In order to achieve full separation in such acase, it is enough to treat the membrane following its pretreatment withlipids. with peptides. This pretreatment in stages offers the advantagethat the achieved effect is reversible and that the duration ofpretreatment (especially as regards to the peptide treatment of thesecond stage) is reduced.

Pretreatment with peptides alone leads to the desired effect in thementioned membranes and as a rule this effect will be permanent.

Preferably the lipids shall be unsaturated vegetable oils. Suitableexamples are rapeseed oil, sunflower oil, coconut oil, peanut oil, oliveoil, maize germ oil and linseed oil.

The peptides or mixtures of peptides used in the invention as a rule arepeptides with short chains and molecular weights up to about10,000--preferably up to about 5,000 and especially preferred up toabout 2,000.

The following peptides are preferred in the invention:

(a) peptides, peptide fractions and peptide extracts from raw milk,fresh milk or whey proteins (obtained from raw or fresh milk),

(b) peptides, peptide fractions and peptide extracts obtained from thehydrolysates of raw milk, fresh milk, whey proteins, vegetable proteins(in particular banana proteins), animal proteins (from fish, eggs, meat)or bacterial proteins (especially yeast proteins), including raw milktreated with enzymes (for instance with liposes such as pancreatin andproteases such as fungal protease), per se,

(c) synthetic peptides, and

(d) raw milk.

Peptide fractions which can be isolated from raw milk, and theespecially preferred peptide fractions arbitrarily denoted A, B and Cillustratively can be obtained as follows:

untreated raw milk is subjected to membrane filtration using a 0.1 to0.6 u membrane,

the filtrate so obtained is subjected to a second membrane filtrationusing a membrane with a cutoff at 1,000 molecular weight,

the filtrate so obtained in subjected to a third membrane filtrationusing a membrane with a cutoff at 6-10,000 molecular weight,

the filtrate so obtained is subject to reverse osmosis at an open orclosed membrane,

in the event an open membrane is used, the filtrate so obtained isconcentrated to about 20% of the volume subjected to reverse osmosis, isleft to stand cold, and precipitated solids are filtered off,

the filtrate so obtained is concentrated further and is set, usingabsolute ethanol, to an ethanol content of about 80% and then isfiltered,

the filtrate so obtained is concentrated, the residue is extracted with90 to 95% ethanol, followed by filtration, and the filtrate then isconcentrated,

the residue so obtained is extracted using chloroform or isopropanol,

the extract solution so obtained is concentrated till dry and theresidue is isolated as product A in the form of a nearly colorlesssolid,

in the event of using a closed membrane, the retentate (in lieu of thefiltrate obtained when using the open membrane) is subjected to theabove stated processing steps and thereby the product A is obtained andto produce B and C,

the retentate of the third membrane filtration and/or the retentate IVfrom the reverse osmosis at the open membrane either is/are concentratedin vacuum and extracted with 80% ethanol, or in that by adding absoluteethanol an ethanol content of 80% is set, the undissolved proportionsbeing then filtered off, the ethanol solution being concentrated tilldry and thereby the product C obtained as a colorless substance,

the remaining undissolved components from the ethanol treatment areextracted with 25% aqueous ethanol, the undissolved ingredients arefiltered off, the filtrate is concentrated in vacuum till dry andthereby the product B has been prepared.

The product A is characterized by:

an HPLC band at RT 4.6; column=DEAE-5PW, Waters, Protein Pak, mobilephase: 10% methanol, 0.5 ml/minute;

an HPLC band at RT 26; column J-125 Waters, Protein Pak, mobile phaseKH₂ PO₄ 0.05 m; 0.5 ml/min

HPLC bands at RT 23.3 (weak); 25.3 (strong); 29.5 (average); 30.5(average); 34.4 (weak);

column=I-125, Waters, Protein Pak

mobile phase: KH₂ PO₄ 0.05 m; 0.5 ml/min;

ninhydrin positive reaction;

good solubility in chloroform, isopropanol, ethanol and water.

The substance B is characterized by:

HPLC bands at RT 19-20 (strong); 21.6 (shoulder); 24.3 (average);

column=I-125, Waters, Protein Pak

mobile phase: KH₂ PO₄ 0.05 m; 0.5 ml/min;

good solubility in 20-30% aqueous ethanol,

ninhydrin positive reaction.

The substance C is characterized by:

HPLC bands at RT-21-22 (weak); 23.4 (strong) and 26.5 (strong)[conditions the same as in G-a];

ninhydrin positive reaction;

good solubility in 70 to 90% aqueous ethanol.

The preparation of these peptides is described below in relation to FIG.1 schematically showing the preparation.

The initial material is fresh, untreated raw milk from a domesticanimal, preferably cow milk. It may have been skimmed in conventionalmanner, for instance by centrifuging. However, skimming also may takeplace jointly with the ensuing first membrane filtration describedbelow.

Said raw milk is subjected to a first membrane filtration at amicroporous membrane with pore sizes in the range of 0.1 to 0.6 u,preferably 0.2 u. Advantageously the membrane filtration system shouldallow filtering in tangential flow. Illustratively a polyvinylidenefluoride membrane was found advantageous, such as a GLVP or HVLP"DURAPORE" membrane made by Millipore Co.

In this manner a clear permeate and a thickly flowing retentate areobtained. The permeate (i.e., the filtrate) contains all salts, lactose,amino acids, oligopeptides and poly-peptides of low molecular weights ingenuine, non-denatured form. The retentate contains practically allcasein and fatty components of the milk.

The dry substance of the filtrate (permeate) amounts to about 6% and thenitrogen content of the dry substance is about 1.2%. Dietetic foodstuffsand food additives may be prepared by concentration.

The filtrate from the first membrane filtration is subjected to a secondmembrane filtration at a membrane with cut-off value of 8-10,000molecular weight. The so-called whey proteins remain essentially in theretentate. The filtrate essentially contains NPN compounds, lactose,short fatty acids and a few salts and is fed to a third membrane forfiltration, of which the cut-off is at 1,000 molecular weight. Theretentate arising there is further described below. The filtrate is fedto a reverse osmosis stage. Operation can be either with a so-calledclosed membrane or with a so-called open membrane. An applicable openmembrane illustratively is an HF membrane made by Millipore Co., typeMr-3NF-40. When an open membrane is used, the desired product A shallmainly be in the filtrate.

In a processing, this product A is concentrated to about 20% of thevolume initially present at the reverse osmosis and then is leftstanding at refrigerator temperatures. Solids are formed, and inparticular lactose will crystallize. The solids are evacuated orfiltered out. The mother liquor is concentrated again by a few percentand is set by means of ethanol to an alcohol content of 80%. When cold,another precipitation takes place, and the precipitate is separated byevacuation or filtration. The filtrate so obtained is concentrated untildry and then is extracted with an approximate ten-fold amount of 90 to95% ethanol. The extract so obtained is concentrated till dry, and theresidue obtained is extracted with isopropanol or chloroform. When theextract is concentrated till dry, the product A is obtained in the formof a colorless substance.

The product A is ninhydrin positive, evinces good solubility inchloroform, isopropanol, ethanol and water and chemically andchromatographically behaves like short-chain peptides.

When using a closed membrane for reverse osmosis, the product A isobtained from the retentate, whereas the permeate (filtrate) practicallyconsists solely of water, of a few salts and of slight amounts of NPNcompounds. Said retentate is processed in the same manner as describedabove for the filtrate of the reverse osmosis at the open membrane.However somewhat larger amount of the aforementioned organic solventsare needed because the retentate contains larger quantities of lactoseand salts than the reverse-osmosis filtrate at the open membrane. In themanner already described, the reverse-osmosis retentate with closedmembrane also yields the product A.

Approximately 100 mg of product A are obtained from one liter of milk.

As already mentioned, the product B/C is obtained from the retentate IIIof the third membrane filtration. The retentate is extracted by means ofabout a ten-fold volume of 80% ethanol. The ethanol extract solution isevacuated or filtered out and the residue is washed once more with alittle 80% ethanol. Then an approximately ten-fold amount of 25% aqueousethanol is used for extraction and during evacuation of the extractsolution, another post-rinse takes place briefly. The extract solutionsso obtained are always concentrated in vacuum till dry. In both cases acolorless solid is obtained, which is denoted here as the product B/C.The two fractions can be used separately or being combined.

The substance yielded by the 25% ethanol extract is denoted "B"; thatobtained from the 80% ethanol extract is denoted "C", latter also beingdenoted hereunder at Rt-103.

Not only the products A, B and C may be used in the invention, but alsothe filtrates, extracts and the reverse-osmosis retentates obtainedwithin the scope of the invention.

The described products can be obtained in the same described manner formso-called "fresh" milk (in the sense of the milk regulations).

Obviously, the initial substances also may be whey or the NPN fractionof a milk free of thermal denaturation. In that case the first and resp.the first and second membrane filtrations are eliminated.

These peptides especially preferred in the invention actually are alsopresent in raw milk in minute amounts. Therefore it is possible also tocarry out pretreatment of the membrane with raw milk. However, thisentails significant length of treatment.

Among the protein hydrolysates described above in (b), those inPCT/EP86/00016 (corresponding to U.S. application Ser. No. 918,253) arepreferred.

The remaining protein hydrolysates mentioned under (b) are preparedsimilarly to the method described in PCT/EP86/00016.

The following Figures are shown:

FIG. 1 is a photograph of the filtrates obtained from the Example.

FIG. 2 is a chromatogram (HPLC) of a filtrate obtained according to theinvention.

FIG. 3 is a reproduction of a membrane pretreated in the manner of theinvention, following spraying with ninhydrin, in comparison with theuntreated membrane.

Using the process of the invention, it is possible to separate thedissolved and undissolved constituents of milk (especially in thetangential flow procedure). A clear filtrate (permeate) is obtained anda very viscous retentate. The filtrate contains the dissolvedconstituents, namely all salts, lactose, amino acids and other NPNcomponents and also oligopeptides and low-molecular weight poly-peptides(whey proteins). The filtrate is bacteria-free, casein-free andfat-free, and moreover it contains no calcium- and phosphate-ionsbecause the casein otherwise binding the calcium- and phosphate-ions isnot denatured in the process of the invention.

"Raw milk" and "Fresh milk" in the sense of this invention mean thatfresh, untreated raw milk and fresh milk of domestic animal, preferablycow milk, is used and was subjected to none of convention heattreatments (pasteurization, ultra high heating and sterilization)employed by the dairies. However, the milk may be skimmedconventionally, for instance by centrifuging.

The following Examples elucidate the invention.

EXAMPLE 1

20 liters of fresh raw (cow) milk are subjected to tangential filtrationusing a polyvinylidene fluoride membrane with a pore size of 0.2 u.About 17 liters of filtrate and 3 liters of retentate are obtained. Theretentate essentially consists of caseins and fat.

The filtrate (17 liters), dry substance about 6% and nitrogen content ofthe dry substance about 1.2% is fed to a second membrane filtrationusing a membrane with a cutoff at 6-10,000 molecular weight. About 1/2to 1 liter of retentate are obtained containing mostly whey proteins andenzymes. The filtrate is about 16 liters and is fed to a third membranefiltration using a membrane with a cutoff of 1,000 molecular weight. Inthis case too only 1/2 to 1 liter of retentate are obtained. The productB/C is recovered from that retentate in the manner described furtherbelow.

Next, the filtrate is fed to reverse osmosis. An open membrane is used,the filtrate so obtained is concentrated to about 3 liters and left tostand for several hours in the refrigerator. A few products, inparticular lactose, crystallize. The solids are filtered, the filtrateis concentrated until, by adding an approximately ten-fold volume ofabsolute ethanol, an ethanol content of about 80% results in the totalmixture. The mixture is thoroughly agitated, then filtration orevacuation takes place regarding the undissolved components. Thefiltrate is concentrated till dry. The residue so obtained is extractedwith 90-95% ethanol (from about ten-fold volume). The extract solutionis filtered off or evacuated and concentrated. The residue is treatedwith an approximately ten-fold amount of chloroform or with anapproximately ten-fold amount of isopropanol. The solution is evaporatedand desired product A is obtained.

If a closed membrane is used in reverse osmosis, then the desiredsubstance shall be in the retentate which then must be processed in themanner described above for the filtrate.

To prepare the product B/C, the retentate III from the third membranefiltration is mixed with about the five-fold amount of absolute ethanolresulting in a mixture being about 80% ethanol. This mixture is stirredintensively and allowed to stand for several hours, after which it isfiltered from the undissolved constituents and concentrated till dry.Next the residue from the extractive treatment is post-treated with 25%aqueous ethanol (from the approximately ten-fold volume). Evacuation orfiltration is carried out regarding the undissolved components, thefiltrate is concentrated till dry and a second fraction of the productB/C s obtained. The two products may be combined.

EXAMPLE 2

A polyvinylidene fluoride membrane with a pore side of 0.2 u (DURAPORE,made by Millipore Co.), was cleaned and rinsed for an hour with water.The membrane fails to filter the milk clear.

Thereupon, the membrane is treated by means of 0.1% solution of fractionA (prepared in the manner of EXAMPLE 1) for 8 and for 14 h withcontinuous flow and at room temperature. Thereupon, the membrane issuitable to separate the dissolved and undissolved constituents of themilk. A clear filtrate is obtained. The properties of the membraneremain unchanged even after treatment with 0.3N NaOH.

The same results will be obtained when using a 0.6 u membrane.

EXAMPLE 3

A DURAPORE membrane (0.2 u) is cleaned and rinsed as described inEXAMPLE 2. Thereupon the membrane is treated in continuous flow for 3.5h at room temperature with a 0.3% solution of Rt-103-25 (prepared perEXAMPLE 1). Following this pre-treatment, the membrane is suitable toseparate the dissolved and undissolved constituents of the milk; a clearfiltrate is obtained. A one-hour treatment of the pretreated membranewith 0.6% NaOH does not affect the properties of the membrane.

The same results are obtained when using a membrane with a 1 u poresize.

EXAMPLE 4

Four 0.2 u DURAPORE membranes were treated as follows:

Membrane 1: was cleaned and rinsed as in EXAMPLE 2;

Membrane 2: the cleaned membrane was treated for 2 h at room temperaturewith a 4% emulsion of rapeseed oil in water and continuous flow;

Membrane 3: the cleaned membrane was treated 8 h at room temperaturewith a 4% aqueous rapeseed oil emulsion in continuous flow;

Membrane 4: was treated first like Membrane 3 and then 2 h with an 8%total hydrolysate from raw milk.

By means of these membranes the attempt was made to separate thedissolved and undissolved constituents of milk. The filtrates soobtained are shown in FIG. 1. It is clear that the Membrane 1 allows noseparation, while some separation is made possible by Membrane 2,Membrane 3 allows almost complete separation and Membrane 4 providesclear separation of the dissolved and undissolved constituents.

EXAMPLE 5

A 0.1 u polyvinylidene fluoride membrane (DURAPORE) was treated asdescribed in EXAMPLE 2 and so cleaned. Next the membrane is treated 8 hin continuous flow with a 4% emulsion of sunflower oil in water, at roomtemperature. Thereupon the membrane was suitable to filter milk clear.

EXAMPLE 6

A 0.6 u polyvinylidene fluoride membrane (DURAPORE) was treated incontinuous flow at room temperature with an 8% raw-milk totalhydrolysate. After 8 h the milk could be filtered clear.

If the same membrane is treated 31/2 h with a 0.5% solution of Rt-103-25(made as in EXAMPLE 1), the same result is obtained.

If the membrane so pretreated is sprayed with ninhydrin reagent, bluecoloration appears on the pretreated part of the membrane denoting thepresence of peptide nitrogen (FIG. 3). The untreated parts of themembrane lack coloration (white strips in FIG. 3).

As mentioned, the filtrate obtained in the manner of the inventioncontains the dissolved constituents of the milk, in particular the wheyproteins. FIG. 2 illustratively shows the composition of the filtrateobtained in the invention. The chromatogram was obtained under thefollowing conditions.

Column: J-125 Waters, Protein Pak

Mobile phase: 10% methanol; 0.5 ml/minute;

Extinction: 0 to 0.2; 224 nm;

Sample: 20 ul.

I claim:
 1. A process for separating dissolved and undissolvedconstituents of milk comprising pretreating a polyvinylidene fluoridemembrane with a pore size in the range of from 0.1 to 2 microns byexposing it to a flowing aqueous solution, dispersion or emulsion oflipids or peptides or a mixture thereof and separating said dissolvedand undissolved constituents of milk by passing the milk through saidpretreated membrane.
 2. The process defined by claim 1, wherein saidmembrane has a pore size in the range of 0.1 to 1 micron.
 3. The processdefined by claim 2, wherein said pore size is in the range of 0.1 to 0.6micron.
 4. The process defined by claim 1, wherein said membrane isinitially pretreated by exposing it to an aqueous solution, dispersionor emulsion of lipids and then pretreated by exposing it to an aqueoussolution, dispersion or emulsion of peptides.
 5. The process defined byclaim 1, wherein said lipids are unsaturated vegetable oils.
 6. Theprocess defined by claim 5, wherein said lipids are members selectedfrom the group consisting of rapeseed oil, sunflower oil, coconut oil,peanut oil, olive oil, maize germ oil and linseed oil.
 7. The processdefined by claim 1, wherein said peptides are members selected from thegroup consisting of peptides, peptide fractions and peptide extractsobtained from raw milk, or whey obtained from raw milk.
 8. The processdefined by claim 1, wherein said peptides are members selected from thegroup consisting of peptides, peptide fractions and peptide extractsobtained from the hydrolysates of raw milk, whey proteins, vegetableproteins, other animal proteins or bacterial proteins and enzyme-treatedmilk.
 9. The process defined by claim 1, wherein said peptides aresynthetic peptides.
 10. The process defined by claim 1, wherein saidpeptides come from raw milk.
 11. The process defined by claim 1, whereinsaid peptides are prepared by a process comprising:subjecting untreatedraw milk to membrane filtration using a 0.1 to 0.6 micron membrane toproduce a first filtrate, subjecting said first filtrate to a secondmembrane filtration using a membrane with a cutoff at 6-10,000 molecularweight to produce a second filtrate, subjecting said second filtrate toa third membrane filtration using a membrane with a cutoff at 1,000molecular weight to produce a third filtrate, subjecting said thirdfiltrate to reverse osmosis using an open membrane to produce a fourthfiltrate, concentrating said fourth filtrate to about 20% of the initialvolume for the reverse osmosis, cooling the concentrated filtrate toprecipitate a solid substance, filtering said solid substance to obtaina fifth filtrate, concentrating said fifth filtrate, setting theconcentrated filtrate with absolute ethanol to an ethanol content ofabout 80% and filtering the concentrated filtrate to produce a sixthfiltrate, concentrating said sixth filtrate, extracting the concentratedfiltrate with 90 to 95% ethanol and filtering the extracted concentratedfiltrate to produce a seventh filtrate, concentrating said seventhfiltrate to produce a residue and extracting the residue with chloroformor isopropanol to produce an extract solution, concentrating saidextract solution until dry and isolating the residue in the form of anearly colorless solid substance.
 12. The process defined by claim 1,wherein said peptides are prepared by a process comprising:subjectinguntreated raw milk to membrane filtration using a 0.1 to 0.6 micronmembrane to produce a first filtrate, subjecting said first filtrate toa second membrane filtration using a membrane with a cutoff at 6-10,000molecular weight to produce a second filtrate, subjecting said secondfiltrate to a third membrane filtration using a membrane with a cutoffat 1,000 molecular weight to produce a third filtrate, concentratingsaid third filtrate in vacuum and extracting the concentrated filtratewith ethanol at an ethanol content of 80% to produce an ethanolsolution, concentrating said ethanol solution until dry to produce acolorless substance.
 13. A microporous membrane prepared by a processcomprising treating a polyvinylidene fluoride membrane with a pore sizein the range of from 0.1 to 2 microns by exposing it to a flowingaqueous solution, dispersion or emulsion of lipids or peptides or amixture thereof.